Kuniko Matsuda

MiR-134/487b/655 Cluster Regulates TGF-β-induced Epithelial-Mesenchymal Transition and Drug Resistance to Gefitinib by Targeting MAGI2 in Lung Adenocarcinoma Cells.

Epithelial–mesenchymal transition (EMT) has recently been recognized as a key element of cell invasion, migration, metastasis, and drug resistance in several types of cancer, including non–small cell lung cancer (NSCLC). Our aim was to clarify microRNA (miRNA)-related mechanisms underlying EMT followed by acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) in NSCLC. miRNA expression profiles were examined before and after transforming growth factor β1 (TGF-β1) exposure in four human adenocarcinoma cell lines with or without EMT. Correlation between expressions of EMT-related miRNAs and resistance to EGFR-TKI gefitinib was evaluated. miRNA array and real-time quantitative reverse transcription PCR (qRT-PCR) revealed that TGF-β1 significantly induced overexpression of miR-134, miR-487b, and miR-655, which belong to the same cluster located on chromosome 14q32, in lung adenocarcinoma cells with EMT. MAGI2 (membrane-associated guanylate kinase, WW, and PDZ domain–containing protein 2), a predicted target of these miRNAs and a scaffold protein required for PTEN, was diminished in A549 cells with EMT after the TGF-β1 stimulation. Overexpression of miR-134 and miR-487b promoted the EMT phenomenon and affected the drug resistance to gefitinib, whereas knockdown of these miRNAs inhibited the EMT process and reversed TGF-β1–induced resistance to gefitinib. Our study demonstrated that the miR-134/487b/655 cluster contributed to the TGF-β1–induced EMT phenomenon and affected the resistance to gefitinib by directly targeting MAGI2, in which suppression subsequently caused loss of PTEN stability in lung cancer cells. The miR-134/miR-487b/miR-655 cluster may be a new therapeutic target in patients with advanced lung adenocarcinoma, depending on the EMT phenomenon. Mol Cancer Ther; 13(2); 444–53. ©2013 AACR.

Antitumor activity of histone deacetylase inhibitors in non-small cell lung cancer cells: development of a molecular predictive model

To ascertain the potential for histone deacetylase (HDAC) inhibitor-based treatment in non-small cell lung cancer (NSCLC), we analyzed the antitumor effects of trichostatin A (TSA) and suberoylanilide hydroxamic acid (vorinostat) in a panel of 16 NSCLC cell lines via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. TSA and vorinostat both displayed strong antitumor activities in 50% of NSCLC cell lines, suggesting the need for the use of predictive markers to select patients receiving this treatment. There was a strong correlation between the responsiveness to TSA and vorinostat (P < 0.0001). To identify a molecular model of sensitivity to HDAC inhibitor treatment in NSCLC, we conducted a gene expression profiling study using cDNA arrays on the same set of cell lines and related the cytotoxic activity of TSA to corresponding gene expression pattern using a modified National Cancer Institute program. In addition, pathway analysis was done with Pathway Architect software. We used nine genes, which were identified by gene-drug sensitivity correlation and pathway analysis, to build a support vector machine algorithm model by which sensitive cell lines were distinguished from resistant cell lines. The prediction performance of the support vector machine model was validated by an additional nine cell lines, resulting in a prediction value of 100% with respect to determining response to TSA and vorinostat. Our results suggested that (a) HDAC inhibitors may be promising anticancer drugs to NSCLC and (b) the nine-gene classifier is useful in predicting drug sensitivity to HDAC inhibitors and may contribute to achieving individualized therapy for NSCLC patients. [Mol Cancer Ther 2008;7(7):1923–30]

EM703 improves bleomycin-induced pulmonary fibrosis in mice by the inhibition of TGF-β signaling in lung fibroblasts

BackgroundFourteen-membered ring macrolides have been effective in reducing chronic airway inflammation and also preventing lung injury and fibrosis in bleomycin-challenged mice via anti-inflammatory effects. EM703 is a new derivative of erythromycin (EM) without the bactericidal effects. We investigated the anti-inflammatory and antifibrotic effects of EM703 in an experimental model of bleomycin-induced lung injury and subsequent fibrosis in mice.MethodsSeven-week-old male ICR mice were used. All experiments used eight mice/group, unless otherwise noted in the figure legends. Bleomycin was administered intravenously to the mice on day 0. EM703 was orally administered daily to mice. All groups were examined for cell populations in the bronchoalveolar lavage (BAL) fluid and for induction of messenger RNA (mRNA) of Smad3 and Smad4 in the lung tissues by reverse transcriptase (RT)-polymerase chainreaction (PCR) on day 7. Fibroblastic foci were assessed histologically, and the hydroxyproline content was chemically determined in the lung tissues on day 28. We performed assay of proliferation and soluble collagen production, and examined the induction of mRNA of Smad3 and Smad4 by RT-PCR in murine lung fibroblast cell line MLg2908. We also examined Smad3, Smad4 and phosphorylated Smad2/3 (p-Smad2/3) protein assay by western blotting in MLg2908.ResultsBleomycin-induced lung fibrosis, and the infiltration of macrophages and neutrophils into the airspace were inhibited by EM703. The expression of Smad3 and Smad4 mRNA was clearly attenuated by bleomycin, but was recovered by EM703. EM703 also inhibited fibroblast proliferation and the collagen production in lung fibroblasts induced by Transforming growth factor-beta (TGF-β). The expression of Smad3 and Smad4 mRNA in murine lung fibroblasts disappeared due to TGF-β, but was recovered by EM703. EM703 inhibited the expression of p-Smad2/3 and Smad4 protein in murine lung fibroblasts induced by TGF-β.ConclusionThese findings suggest that EM703 improves bleomycin-induced pulmonary fibrosis in mice by actions of anti-inflammation and regulation of TGF-β signaling in lung fibroblasts.

Gefitinib (IRESSA) sensitive lung cancer cell lines show phosphorylation of Akt without ligand stimulation

BackgroundPhase III trials evaluating the efficacy of gefitinib (IRESSA) in non-small cell lung cancer (NSCLC) lend support to the need for improved patient selection in terms of gefitinib use. Mutation of the epidermal growth factor receptor (EGFR) gene is reported to be associated with clinical responsiveness to gefitinib. However, gefitinib-sensitive and prolonged stable-disease-defined tumors without EGFR gene mutation have also been reported.MethodsTo identify other key factors involved in gefitinib sensitivity, we analyzed the protein expression of molecules within the EGFR family, PI3K-Akt and Ras/MEK/Erk pathways and examined the sensitivity to gefitinib using the MTT cell proliferation assay in 23 lung cancer cell lines.ResultsWe identified one highly sensitive cell line (PC9), eight cell lines displaying intermediate-sensitivity, and 14 resistant cell lines. Only PC9 and PC14 (intermediate-sensitivity) displayed an EGFR gene mutation including amplification. Eight out of the nine cell lines showing sensitivity had Akt phosphorylation without ligand stimulation, while only three out of the 14 resistant lines displayed this characteristic (P = 0.0059). Furthermore, the ratio of phosphor-Akt/total Akt in sensitive cells was higher than that observed in resistant cells (P = 0.0016). Akt phosphorylation was partially inhibited by gefitinib in all sensitive cell lines.ConclusionThese results suggest that Akt phosphorylation without ligand stimulation may play a key signaling role in gefitinib sensitivity, especially intermediate-sensitivity. In addition, expression analyses of the EGFR family, EGFR gene mutation, and FISH (fluorescence in situ hybridization) analyses showed that the phosphorylated state of EGFR and Akt might be a useful clinical marker of Akt activation without ligand stimulation, in addition to EGFR gene mutation and amplification, particularly in adenocarcinomas.

Anticancer drug clustering in lung cancer based on gene expression profiles and sensitivity database

AbstractbackgroundThe effect of current therapies in improving the survival of lung cancer patients remains far from satisfactory. It is consequently desirable to find more appropriate therapeutic opportunities based on informed insights. A molecular pharmacological analysis was undertaken to design an improved chemotherapeutic strategy for advanced lung cancer.MethodsWe related the cytotoxic activity of each of commonly used anti-cancer agents (docetaxel, paclitaxel, gemcitabine, vinorelbine, 5-FU, SN38, cisplatin (CDDP), and carboplatin (CBDCA)) to corresponding expression pattern in each of the cell lines using a modified NCI program.ResultsWe performed gene expression analysis in lung cancer cell lines using cDNA filter and high-density oligonucleotide arrays. We also examined the sensitivity of these cell lines to these drugs via MTT assay. To obtain our reproducible gene-drug sensitivity correlation data, we separately analyzed two sets of lung cancer cell lines, namely 10 and 19. In our gene-drug correlation analyses, gemcitabine consistently belonged to an isolated cluster in a reproducible fashion. On the other hand, docetaxel, paclitaxel, 5-FU, SN-38, CBDCA and CDDP were gathered together into one large cluster.ConclusionThese results suggest that chemotherapy regimens including gemcitabine should be evaluated in second-line chemotherapy in cases where the first-line chemotherapy did not include this drug. Gene expression-drug sensitivity correlations, as provided by the NCI program, may yield improved therapeutic options for treatment of specific tumor types.

Reduction in serum high mobility group box-1 level by polymyxin B-immobilized fiber column in patients with idiopathic pulmonary fibrosis with acute exacerbation.

Background/Aim: Recent reports suggest that polymyxin B (PMX)-immobilized fiber may have beneficial effects in idiopathic pulmonary fibrosis (IPF) with acute exacerbation (AE). High mobility group box-1 (HMGB-1) is an important pro-inflammatory mediator that contributes to acute lung inflammation. This study was aimed to investigate whether PMX treatment affects serum HMGB-1 levels and oxygenation in IPF patients with AE. Materials and Methods: Twenty IPF patients with AE were treated by PMX. PMX treatment was carried out once daily for 2 successive days. Serum HMGB-1 levels were measured before and after PMX treatment. We also monitored arterial oxygen tension (PaO2)/inspiratory oxygen fraction (FiO2) (P/F) ratio. PMX fiber columns were analyzed to examine whether HMGB-1 was absorbed by PMX. Results: PMX treatment significantly improved both the serum HMGB-1 level and P/F ratio. HMGB-1 was detected in washing medium from the PMX column. Conclusion: PMX treatment may reduce serum HMGB-1 and improve oxygenation in patients with IPF with AE.

Neutrophil Adsorption by Polymyxin B-Immobilized Fiber Column for Acute Exacerbation in Patients with Interstitial Pneumonia: A Pilot Study

Background/Aims: Polymyxin B-immobilized fiber (PMX) treatment has beneficial effects in patients with acute lung injury/acute respiratory distress syndrome or acute exacerbation of idiopathic pulmonary fibrosis. This study was aimed to clarify the mechanism of PMX treatment for acute exacerbation of interstitial pneumonia (IP). Materials and Methods: Sixteen consecutive IP patients with acute exacerbation were included. The patients were treated with PMX once daily for 2 successive days at a flow rate of 80–100 ml/min for 6 h. Cells adsorbed by PMX were analyzed morphologically by electron microscopy. Surface markers of these cells were determined by flow cytometry. Serum matrix metalloproteinase (MMP)-9 was measured before and after PMX treatment. Results: Cells adsorbed by PMX were neutrophils and highly expressed HLA-DR, CD14, CD62L and CD114. Serum MMP-9 levels were significantly decreased after PMX treatment. Conclusion: This pilot study demonstrated neutrophil adsorption by PMX and its possible clinical application for acute exacerbation of IP.

Pirfenidone inhibits fibrocyte accumulation in the lungs in bleomycin-induced murine pulmonary fibrosis

BackgroundBone marrow-derived fibrocytes reportedly play important roles in the pathogenesis of idiopathic pulmonary fibrosis. Pirfenidone is an anti-fibrotic agent; however, its effects on fibrocytes have not been investigated. The aim of this study was to investigate whether pirfenidone inhibits fibrocyte pool size in the lungs of bleomycin-treated mice.MethodsBleomycin (100 mg/kg) was infused with osmotic pumps into C57BL/6 mice, and pirfenidone (300 mg/kg/day) was orally administered daily for 2 wk. The lungs were removed, and single-cell suspensions were subjected to fluorescence-activated cell sorter (FACS) analysis to detect fibrocytes, which were defined as CD45 and collagen-I double-positive cells. Immunohistochemistry was performed on the lung specimens to quantify fibrocytes. Chemokines in the lung digests were measured with enzyme-linked immunosorbent assay. The effect of pirfenidone on alveolar macrophages was evaluated with bronchoalveolar lavage (BAL). In a therapeutic setting, pirfenidone administration was initiated 10 days after bleomycin treatment. For chemotaxis assay, lung fibrocytes were isolated with immunomagnetic selection (CD45-positive mesenchymal cells) after culture and allowed to migrate toward chemokines in the presence or absence of pirfenidone. Moreover, the effect of pirfenidone on the expression of chemokine receptors on fibrocytes was evaluated.ResultsPirfenidone significantly ameliorated bleomycin-induced pulmonary fibrosis as assessed with quantitative histology and collagen measurement. Fibrocyte pool size in bleomycin-treated mice lungs was attenuated from 26.5% to 13.7% by pirfenidone on FACS analysis. This outcome was also observed in a therapeutic setting. Immunohistochemistry revealed that fibrocytes were significantly decreased by pirfenidone administration compared with those in bleomycin-treated mice (P = 0.0097). Increased chemokine (CC motif) ligand-2 (CCL2) and CCL12 production in bleomycin-treated mouse lungs was significantly attenuated by pirfenidone (P = 0.0003 and P < 0.0001, respectively). Pirfenidone also attenuated macrophage counts stimulated by bleomycin in BAL fluid. Fibrocyte migration toward CCL2 and chemokine (CC motif) receptor-2 expression on fibrocytes was significantly inhibited by pirfenidone in vitro.ConclusionsPirfenidone attenuated the fibrocyte pool size in bleomycin-treated mouse lungs via attenuation of CCL2 and CCL12 production in vivo, and fibrocyte migration was inhibited by pirfenidone in vitro. Fibrocyte inhibition is considered a mechanism of anti-fibrotic action of pirfenidone.

Histone deacetylase inhibitor enhances sensitivity of non-small-cell lung cancer cells to 5-FU/S-1 via down-regulation of thymidylate synthase expression and up-regulation of p21waf1/cip1 expression

It is desirable to find more appropriate therapeutic opportunities in non‐small‐cell lung cancer (NSCLC) due to the current poor prognosis of affected patients. Recently, several histone deacetylase (HDAC) inhibitors, including suberoylanilide hydroxamic acid (SAHA), have been reported to exhibit antitumor activities against NSCLC. S‐1, a novel oral fluorouracil anticancer drug, has been developed for clinical use in the treatment of NSCLC in Japan. Using an MTT assay, we analyzed the growth‐inhibitory effect of 5‐fluorouracil (5‐FU), S‐1, and SAHA against three NSCLC cell lines, as well as the breast cancer cell line MCF7 which is known to be highly sensitive to 5‐FU. Combined treatment with low‐dose SAHA enhanced 5‐FU‐ and S‐1‐mediated cytotoxicity and resulted in synergistic effects, especially in 5‐FU‐resistant cells. Both the mRNA and protein expression levels of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), and orotate phosphoribosyltransferase (OPRT), which are associated with 5‐FU sensitivity/response, were analyzed in the cells undergoing treatment. 5‐Fluorouracil‐resistant lung cancer cells displayed high expression of TS mRNA and protein. Suberoylanilide hydroxamic acid down‐regulated TS mRNA and protein expression, as well as repressed the rapid induction of this factor during 5‐FU treatment, in all examined cell types. We also examined the status of the Rb‐E2F1 pathway, with SAHA up‐regulating p21waf1/cip1 expression via promoter histone acetylation; this, in turn, blocked the Rb‐E2F1 pathway. We conclude that combination therapy with SAHA and S‐1 in lung cancer may be promising due to its potential to overcome S‐1 resistance via modulation of 5‐FU/S‐1 sensitivity‐associated biomarker (TS) by HDAC inhibitor. (Cancer Sci 2010)

Reduced transcription of the RB2/p130 gene in human lung cancer

Reduced expression of the retinoblastoma gene (RB)2/p130 protein, as well as mutation of exons 19, 20, 21, and 22 of the same gene, has been reported in primary lung cancer. However, it has been suggested by other investigators that mutational inactivation and loss of the RB2/p130 gene and protein, respectively, are rare events in lung cancer. In order to determine the contribution and mechanisms of RB2/p130 gene inactivation to lung cancer development and progression, we quantified RB2/p130 mRNA expression levels in a range of human lung cancer cell lines (n = 13) by real‐time reverse transcription (RT)‐polymerase chain reaction (PCR) analysis. In comparison to normal lung tissue, reduced transcription of the RB2/p130 gene was found in all small cell lung cancer cell lines examined, along with six out of the eight nonsmall cell lung cancers tested, most of which had inactivation of RB/p16 pathway. On the basis of Western blot analysis, the expression of RB2/p130 protein was consistent with RNA expression levels in all lung cancer cell lines examined. In addition, the mutational status of the RB2/p130 gene (specifically, exons 19, 20, 21, and 22) was determined in 30 primary lung cancers (from patients with distant metastasis) and 30 lung cancer cell lines by PCR‐single strand conformation polymorphism (SSCP) analysis and direct DNA sequencing. There was no evidence of somatic mutations within the RB2/p130 gene in the 60 lung cancer samples (both cell lines and tumors) assessed, including the 11 lung cancer cell lines that displayed reduced expression of the gene. Furthermore, hypermethylation of the RB2/p130 promoter was not found in any of the above‐mentioned 11 cell lines, as determined by a DNA methylation assay, combined bisulfite restriction analysis (COBRA). The results of the present study suggest that the reduced RB2/p130 expression seen in lung cancer may be in part transcriptionally mediated, albeit not likely via a mechanism involving hypermethylation of the RB2/p130 promoter. The observed reduction in RB2/p130 gene expression may be due to histone deacetylation, altered mRNA stability, and/or other forms of transcriptional regulation.